Phenylalanine-based inactivator of akt kinase: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1021/ml500088x

Strategies to inhibit kinases by targeting the substrate binding site offer many advantages, including naturally evolved selectivity filters, but normally suffer from poor potency. In this work we propose a strategy to design and prepare covalent substrate-competitive kinase inhibitors as a method to improve potency. We have chosen AKT as the model kinase for this work. Using the AKT-GSK3β cocrystal structure and a reactive cysteine near the substrate binding site, we have identified phenylalanine (Phe) as an appropriate scaffold for the covalent inactivator portion of these inhibitors. By synthesizing compounds that incorporate cysteine-reactive electrophiles into phenylalanine and testing these compounds as AKT inhibitors, we have identified Boc-Phe-vinyl ketone as a submicromolar inactivator of AKT. We also show that Boc-Phe-vinyl ketone (1) potently inhibits AKT1 and inhibits cell growth in HCT116 and H460 cells nearly as well as AKT inhibitors GSK690693 and MK-2206, (2) is selective for kinases that possess an activation loop cysteine such as AKT, (3) requires the vinyl ketone for inactivation, (4) has inactivation that is time-dependent, and (5) alkylates Cys310 of AKT as shown by mass spectrometry. Identification of Boc-Phe-vinyl ketone as a covalent inactivator of AKT will allow the development of peptide and small-molecule substrate-competitive covalent kinase inhibitors that incorporate additional substrate binding elements to increase selectivity and potency. This proof-of-principle study also provides a basis to apply this strategy to other kinases of the AGC and CAMK families.

Full text of DOI:10.1021/ml500088x

Letter
pubs.acs.org/acsmedchemlett
Phenylalanine-Based Inactivator of AKT Kinase: Design, Synthesis,
and Biological Evaluation
Thuy Nguyen,† Robert A. Coover,† Jenson Verghese,† Richard G. Moran,‡ and Keith C. Ellis*,†
†Department of Medicinal Chemistry, School of Pharmacy, and the Massey Cancer Center, Virginia Commonwealth University,
Richmond, Virginia 23298-0540, United States
‡Department of Pharmacology & Toxicology, School of Medicine, and the Massey Cancer Center, Virginia Commonwealth
University, Richmond, Virginia 23298-0540, United States
S
* Supporting Information
ABSTRACT: Strategies to inhibit kinases by targeting the
substrate binding site offer many advantages, including
naturally evolved selectivity filters, but normally suffer from
poor potency. In this work we propose a strategy to design and
prepare covalent substrate-competitive kinase inhibitors as a
method to improve potency. We have chosen AKT as the
model kinase for this work. Using the AKT-GSK3β cocrystal
structure and a reactive cysteine near the substrate binding site, we have identified phenylalanine (Phe) as an appropriate scaffold
for the covalent inactivator portion of these inhibitors. By synthesizing compounds that incorporate cysteine-reactive
electrophiles into phenylalanine and testing these compounds as AKT inhibitors, we have identified Boc-Phe-vinyl ketone as a
submicromolar inactivator of AKT. We also show that Boc-Phe-vinyl ketone (1) potently inhibits AKT1 and inhibits cell growth
in HCT116 and H460 cells nearly as well as AKT inhibitors GSK690693 and MK-2206, (2) is selective for kinases that possess
an activation loop cysteine such as AKT, (3) requires the vinyl ketone for inactivation, (4) has inactivation that is time-
dependent, and (5) alkylates Cys310 of AKT as shown by mass spectrometry. Identification of Boc-Phe-vinyl ketone as a
covalent inactivator of AKT will allow the development of peptide and small-molecule substrate-competitive covalent kinase
inhibitors that incorporate additional substrate binding elements to increase selectivity and potency. This proof-of-principle study
also provides a basis to apply this strategy to other kinases of the AGC and CAMK families.
KEYWORDS: AKT kinase, substrate competitive, covalent inhibitor
o date, the majority of kinase inhibitors that have been
torial libraries of peptides,7,8 inclusion of unnatural amino
Tdeveloped target the ATP binding site. This strategy, acids,9 and bisubstrate approaches linking the substrate peptide
to ATP-competitive scaffolds.10,11
however, has several limitations. The ATP binding site is highly
Here we describe the development of covalent substrate-
competitive kinase inhibitors from the substrate sequence as a
new strategy to increase potency. Covalent inhibitors are
known to have several advantages including increased
biochemical efficiency due to irreversible binding, longer
duration of action, increased efficacy achieved at lower drug
concentrations, and the potential to avoid some drug resistance
mechanisms.12,13 Despite the known drawbacks of lack of
specificity and potential for immunogenic reaction to the
protein−inhibitor adduct, there has been renewed and
increasing interest in covalent inhibitors. The strategy reported
here can potentially provide rapid access to inhibitors for
kinases for which no inhibitor is currently known, derived solely
from the structure of the kinase and known substrate
sequences.
conserved across the kinome, making selectivity difficult to
achieve.1 ATP-competitive inhibitors can also bind to and
modulate the activity of the ∼1500 nonkinase enzymes that
utilize ATP.2,3 Finally, ATP-competitive inhibitors must
compete with high levels of intracellular ATP (∼2−10 mM)
to be effective.4
Substrate-competitive inhibitors offer many advantages over
ATP-competitive inhibitors.5 The substrate binding site of
kinases has evolved to recognize only specific substrate
sequences.6,7 This natural selectivity filter allows a given kinase
to recognize a limited number of substrates, an important
feature for the control of cellular signaling. These substrates are
typically present in the cell in much lower concentration than
ATP. Taken together, these factors would highly recommend a
substrate-competitive approach to kinase inhibitor develop-
ment. However, while substrate peptides have high specificity
for a given kinase, these sequences themselves are usually poor
inhibitors due to lack of potency.5
To develop this strategy, we have chosen AKT as a model
because detailed knowledge of the structural interactions
Various strategies have been used to increase the potency of
substrate-competitive inhibitors derived from natural substrate
sequences including the synthesis and screening of combina-
Received: August 29, 2013
Accepted: March 7, 2014
Published: March 7, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
between the kinase and a fragment of its GSK3β substrate are
available.14 Our approach to combine the substrate peptide
sequence with an electrophile to produce a covalent inhibitor
(Figure 1a) requires that we (1) identify a reactive amino acid
in the kinase and (2) identify the amino acid in the substrate
that binds in close proximity.
The syntheses of compounds based on the phenylalanine are
shown in Scheme 1. Starting from commercially available Boc-
a
Scheme 1. Synthesis of 3, 5−7, and 8
a
Reagents and conditions: (a) MeON(H)Me·HCl, HOBt, EDCI,
DCM, NMM, 0 °C to rt, overnight, 92%; (b) vinylmagnesium
bromide, THF, −40 °C, 3 h, 70%; (c) ethanolamine, HOBt, EDCI,
DCM, NMM, 0 °C, 3 h, 90%; (d) for 5, I2, PPh3, imid., DCM, rt, 15
min, 51%; for 6, CBr4, PPh3, DCM, 0 °C, 2 h, 50%; for 7, CCl4, PPh3,
DCM, 0 °C, 2 h, 50%; (e) maleimide, DEAD, PPh3, DCM, −78 °C to
rt, overnight, 44%.
Phe-OH 1, we prepared Weinreb amide 2 by N-(3-
dimethylaminopropyl)-N′-ethylcarbo-diimide (EDC) coupling
and converted it to the vinyl ketone 3 by treatment with
vinylmagnesium bromide. Also starting from Boc-Phe-OH 1,
we prepared amide 4 by EDC coupling. The primary alcohol of
4 was then converted to the halides 5−7 using triphenylphos-
phine and the appropriate halogen source. Alcohol 4 was also
used to prepare maleimide 8 by Mitsunobu reaction.
Figure 1. (a) Strategy for peptide-based substrate-competitive covalent
inhibitors of AKT. (b) Co-crystal structure of AKT with a ten amino
acid fragment of the substrate GSK3β (PDB ID: 1O6K).14 (c) General
design of phenylalanaine-based covalent inactivators of AKT.
To explore other electrophiles, such as α-halo-acetamides
and the vinyl amide, and to vary the linker length of the
maleimide, we prepared aza-phenylalanine analogues where the
Cα has been replaced by nitrogen (Schemes 2 and 3). Starting
from commercially available N-Boc-hydrazine, reductive
Several compounds have been reported to covalently modify
Cys310 of the AKT activation loop. These include the
pyranonaphthoquinone (PNQ) lactone natural products,15−17
the endogenous α,β-unsaturated aldehyde 4-hydroynonenal (4-
HNE),18 and N-ethylmaleimide.19 Alkylation of Cys310 of
AKT by the Michael acceptor in the PNQ lactone and 4-HNE
has been demonstrated by mass spectrometry.17,18
a
Scheme 2. Synthesis of 11−14
The cocrystal structure of AKT with a ten amino acid
fragment of the substrate GSK3β has been reported (PDB ID:
1O6K).14 As can be seen in Figure 1b, Cys310 is located on the
C-terminal end of the AKT activation loop and is located
adjacent to the substrate binding site. Phe10 of the GSK3β
peptide binds in close proximity to Cys310. The Cα to Cα
distance between these two residues is ∼5.5 Å. On the basis of
this proximity, we chose phenylalanine as the scaffold for our
covalent inactivator of AKT.
As the first step in a program to develop peptide-based
substrate-competitive covalent inhibitors of AKT, we have
designed, synthesized, and evaluated a set of phenylalanine
derivatives that contain a C-terminal cysteine-reactive electro-
phile. The general design of these inactivators is shown in
Figure 1c. The electrophiles that we evaluated in this set of
compounds include a vinyl ketone, alkyl halides, maleimides, α-
halo acetamides, and a vinyl amide.
a
Reagents and conditions: (a) i. PhCHO, THF, rt, 4 h; ii. NaBH3CN,
AcOH, THF, 0 °C to rt, overnight; iii. NaOH, MeOH, rt, 2h, 82%; (b)
for 11, chloroacetyl chloride, Et3N, THF, 0 °C to rt, overnight, 93%;
for 12, bromoacetyl chloride, Et3N, THF, 0 °C to rt, overnight, 95%;
(c) NaI, acetone, reflux, overnight, 86%; (d) acryloyl chloride, Et3N,
THF, 0 °C to rt, overnight, quantitative.
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a
ketone 17 (Scheme 4). Testing of 17 for inhibition of AKT1 in
the Z′-LYTE assay showed that this compound is inactive, with
Scheme 3. Synthesis of 16
a
Scheme 4. Synthesis of Ethyl Ketone 17
a
Reagents and conditions: (a) 15, K2CO3, MeCN, reflux, overnight,
55%; (b) PhOMe, 150 °C, 1.5 h, 62%.
a
Reagents and conditions: (a) Pd/C, H2, EtOAc, rt, overnight,
amination with benzaldehyde afforded N-Boc-N′-benzyl-
hydrazine 10. Treatment of 10 with chloroacetyl chloride or
bromoacetyl chloride afforded α-chloroacetamide 11 and α-
bromoacetamide 12, respectively. α-Chloroacetamide 11 was
then further converted to the α-iodoacetamide 13 under
Finkelstein conditions. Vinyl amide 14 was prepared by
reaction of hydrazine 10 with acryloyl chloride. Finally, α-
iodoacetamide 13 was treated with protected maleimide 15
followed by thermal retro-Diels−Alder deprotection to afford
maleimide 16 (Scheme 3).
quantitative.
an IC50 >100 μM (Table 1). As a second method to determine
whether 3 is a covalent inhibitor, we examined the time-
dependence of its inhibition of AKT1. We retested 3 in the Z′-
LYTE assay, varying the incubation time of the kinase with 3
prior to the kinase reaction step from 0 to 60 min (Figure 2).
The data show a progressive, time-dependent decrease in IC50
values, a characteristic of irreversible inhibitors that interact
covalently with their target enzyme.
The phenylalanine-electrophile analogues prepared in
Schemes 1−3 were tested in the Z′-LYTE assay (Invitrogen)
for their ability to inhibit AKT1 (Table 1). This FRET-based
Table 1. Inhibition of AKT1 by Phenylalanine-Based
a
Covalent Inactivators
b
compd
AKT1 IC50 (μM)
3
0.58 (0.46−0.75)
>100
5
6
>100
7
>100
8
4.48 (3.93−5.10)
>100
11
12
13
14
16
17
24.66 (21.59−28.16)
16.13 (14.64−17.77)
>100
Figure 2. Inhibition of AKT1 by Boc-Phe-vinyl ketone 3 is time-
dependent.
4.68 (3.29−6.65)
>100
GSK690693
MK-2206
0.0006 (0.0003−0.0009)
0.010 (0.006−0.016)
To evaluate the selectivity of Boc-Phe-vinyl ketone 3, we first
tested its ability to inhibit c-AMP-dependent protein kinase A
(PKAα), also in the Z′-LYTE assay. PKAα, like AKT, is a
member of the AGC kinase family and is the most closely
related kinase to AKT, sharing 43% sequence identity in the
kinase catalytic domain.22 Boc-Phe-vinyl ketone 3 inhibited
PKAα with an IC50 of 52.6 μM and is thus ∼90-fold more
selective for AKT1 than PKAα. To further evaluate the
selectivity of 3, we tested its inhibitory effects in a panel of 45
kinases using the SelectScreen Profiling Service from Life
Technologies (see Table S1 in the Supporting Information for
complete data set). This panel of 45 kinases was composed of
22 kinases that contain an activation loop cysteine similar to
AKT (see Table S1, Supporting Information, for sequences and
alignments) and 23 kinases that do not have an activation loop
cysteine. All 45 of the kinases contained at least one cysteine,
with 40 of the kinases containing >5 cysteines and six kinases
containing >30 cysteines (see Table S1, Supporting Informa-
tion, for the number of cysteines per kinase). Of the 45 kinases
tested, only four were inhibited as well or better than AKT:
AMPKα1, CaMKIV, CHK2, and MELK. As can be seen in
Table 2, all four of these kinases contain a cysteine in the same
position of their activation loops as in AKT. Most interestingly,
these kinases all belong to the CAMK family of kinases. Other
a
b
IC50s were determined using Z′-LYTE assay kits (Invitrogen). Data
represents the averages with 95% confidence intervals shown in
parentheses of three independent experiments.
assay measures the ability of a kinase enzyme to phosphorylate
a peptide substrate and detects functional inhibitors of kinase
activity independent of mechanism of action. Alkyl halides 5−7,
α-chloroacetamide 11, and vinyl amide 14 were all inactive as
AKT1 inhibitors, with IC50s of >100 μM. α-Bromoacetamide
12, α-iodoacetamide 13, and maleimides 8 and 16 all inhibited
AKT1 in the low micromolar range of concentration. Boc-Phe-
vinyl ketone 3, however, inhibited AKT1 at submicromolar
concentration, with an IC50 of 580 nM. Known AKT inhibitors
in clinical trials from GlaxoSmithKline (GSK690693) and
Merck (MK-2206) were used as controls. The IC50 values
determined for these compounds in our assay were consistent
with reported values (GSK690693, 2 nM;20 MK-2206, 8 nM21).
Having identified Boc-Phe-vinyl ketone 3 as a potential
candidate for an AKT-selective covalent inactivator, we wished
to confirm that this compound inhibits AKT by a covalent
mechanism of action. As our first experiment, we inactivated
the electrophile of 3 by reducing the vinyl ketone to the ethyl
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Table 2. Kinases Inhibited in SelectScreen Panel
kinase
AKT1
% inhibition @ 1 μM of 3
activation loop cysteine
50
46
50
46
62
308TFCGT312
183TSCGS187
200TVCGT204
383TLCGT387
167TCCGS171
AMPKα1
CaMKIV
CHK2
MELK
kinases of the AGC family that carry an activation loop cysteine
similar to AKT (such as PKAα, PKCα, PKCθ, PKG1, RSK1,
MSK1, and p70S6K) were not inhibited by 3. We are currently
analyzing the consensus sequences for AMPKα1, CaMKIV,
CHK2, and MELK to generate hypotheses for binding sites and
potential use of 3 as an inactivator of these kinases (see
Supplemental Discussion in the Supporting Information). Also
of note, the other 18 kinases tested that carry the activation
loop cysteine similar to AKT and all 23 of the kinases that
contained cysteines in the protein but not specifically in the
activation loop were not inhibited by 3 (see Table S1 in the
Supporting Information). Taken together this data demon-
strates that 3 selectively inhibits only a subset of kinases that
contain the activation loop cysteine and does not inhibit the
function of kinases lacking the activation loop cysteine.
To determine if inhibition of AKT by Boc-Phe-vinyl ketone 3
is effective in a cellular context, we performed growth inhibition
assays in HCT116 (colon cancer) and H460 (nonsmall cell
lung cancer) cell lines. Both of these cell lines carry genetic
mutations that drive AKT signaling by constitutively activating
the PI3K/AKT signaling pathway. One mutation occurs in the
large subunit of PI3K and one mutation constitutively activates
KRas.23 Selective inhibition of AKT in cancer cell lines has been
shown to inhibit growth.20,24 Known AKT inhibitors MK-2206
and GSK690693 were again used as control compounds. Boc-
Phe-vinyl ketone 3 was moderately potent in the growth
inhibition assay (Table 3 and Figure 3) and the IC50 values for
Figure 3. Growth inhibition of HCT116 colon cancer cells (a) and
H460 nonsmall cell lung cancer cells (b) by Boc-Phe-vinyl ketone 3,
MK-2206, GSK690693, and Boc-Phe-ethyl ketone 17.
Table 3. Growth Inhibition of HCT116 and H460 Cancer
Cell Lines by AKT Inhibitors
a
IC50 (μM)
compd
HCT116 cells
H460 cells
22.9 6.4
as those in Table 2) or enzymes that have not yet been
identified. In addition, the cellular data provide evidence that
Boc-Phe-vinyl ketone 3 is cell permeable. Our hypothesis is that
the compound is actively transported by amino acid trans-
porters, which might account for this increase in inhibition
efficiency. Finally, as expected, the Boc-Phe-ethyl ketone 17
was inactive in both cells lines (IC50 > 100 μM; Table 3 and
Figure 3). These data are in agreement with the inhibition data
with recombinant AKT1 and demonstrates that the vinyl
ketone Michael acceptor is required for inhibition.
Lastly, we determined the amino acid of AKT1 that is
covalently modified by Boc-Phe-vinyl ketone 3 using mass
spectrometry analysis. For this experiment, we used 18, a
fluorescein-labeled analogue of 3 (Figure 4), in order to
confirm protein labeling prior to mass spectrometry analysis.
Incubation of AKT1 with 18 was followed by gel eletrophoresis,
confirmation of labeling by in-gel fluorescence scanning (see
Figure S1 in the Supporting Information), excision of the
labeled protein band, and trypsin digest. Using a LTQ Orbitrap
Velos mass spectrometer, an average of 93% coverage was
obtained. Phe-vinyl ketone 18 (788.26 amu) was shown to
modify the AKT fragment containing Cys310 (Table 4).
3
21.5 4.0
8.2 0.5
7.5 2.3
>100
b
GSK690693
MK-2206
17
5.4 2.2
5.4 3.1
>100
a
b
See Supporting Information for details. n = 4.
3 were within 2- to 4-fold of the known AKT inhibitors in the
HCT116 and H460 cell lines. Interestingly, the ratio of the
cellular IC50 to the IC50 with recombinant AKT is much smaller
with Boc-Phe-vinyl ketone 3 (ratio of 37 for HCT116 and 39
for H460) than for with GSK690693 (ratio of 4100 for
HCT116 and 2700 for H460) or MK-2206 (ratio of 750 for
HCT116 and 540 for H460), demonstrating that 3 is more
efficient in inhibiting growth in these cell lines. This ability to
inhibit cell growth at a moderately potent concentration in cells
while only having a moderately potent IC50 against
recombinant enzyme could be the result of several factors
including the increased biochemical efficiency due to
irreversible binding of the covalent mechanism of action,
increased cell permeability relative to MK-2206 and
GSK690693, or inhibition of off-target cellular enzymes (such
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conditions for these three inhibitors). Boc-Phe-vinyl ketone 3,
however, is quite a small compound (MW = 276), and its
selectivity and potency can likely be improved by adding
additional structural elements from the GSK3β substrate
peptide or small molecule fragments that bind in a similar
fashion. This work is currently underway in our laboratory.
The activation-loop cysteine targeted by the design strategy
in this work is conserved in kinases in the AGC family
(including PKA, PKC, MSK, RSK, S6K, and SGK)26 and
CAMK family (AMPKα1, CaMKIV, CHK2, and MELK). We
are currently working to develop covalent inactivators and
substrate-competitive covalent inhibitors of these enzymes
using this conserved cysteine and their unique substrate
consensus sequences.
Figure 4. Fluorescein-labeled Phe-vinyl ketone 18 used for mass
spectrometry experiment (see Supporting Information).
Modifications of other amino acids in AKT, including Cys296,
were not observed.
Table 4. Mass Spectrometry Data for AKT1 Fragment
a
Modified by 18
308TFCGTPEYLAPEVLEDNDYGR328
ASSOCIATED CONTENT
■
mass found
mass calculated
S
* Supporting Information
unmodified
2389.04
3177.33
2389.06
3177.32
Experimental data for the synthesis and characterization of all
compounds, SelectScreen kinase panel data and supplementary
discussion, assay protocols for inhibition of AKT and PKA, cell
growth inhibition, time-dependence experiments, mass spec-
trometry experiments, and comparison of assay conditions for
noncovalent substrate-competitive inhibitors of AKT. This
material is available free of charge via the Internet at http://
pubs.acs.org.
b
modified with 18
a
Experimental details for the mass spectrometry experiments can be
found in the Supporting Information. Two new ions (+3 and +4) were
found from the modified peptide with an unambiguous MS/MS
sequence. Exact mass of Boc-Phe-vinyl ketone 18 is 788.26 amu
b
(calculated).
Strategies to develop substrate-competitive kinase inhibitors
that disrupt protein−protein interactions are highly sought
because they offer the potential of selectivity for a specific
kinase. This selectivity is derived from utilizing the same
structural elements as Nature for binding. One method to
improve the poor potency of substrate-competitive kinase
inhibitors is to develop ones that act by a covalent mechanism
of action. As a first step in this process, we have designed and
synthesized a covalent inactivator of AKT, Boc-Phe-vinyl
ketone 3, and shown that is it potent (against recombinant
enzyme and in a cell growth inhibition assay), selective (for
AKT and a subset of kinases that contain an activation loop
cysteine), and selectively labels Cys199 as designed.
In comparison to known noncovalent substrate-competitive
inhibitors of AKT, Boc-Phe-vinyl ketone 3 is more potent than
the small-molecule inhibitor reported by Hamilton (IC50 = 12
μM;25 Figure 5) but less potent that the heptapeptide reported
by Livnah (IC50 = 0.2 μM;9 Figure 5) (see Table S3 in the
Supporting Information for a comparison of the assay
AUTHOR INFORMATION
■
Corresponding Author
*(K.C.E.) Tel: 804-828-4490. E-mail: kcellis@vcu.edu.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This research was supported by Institutional Research Grant
IRG-73-001-37 from the American Cancer Society (to K.C.E,
administered by the VCU Massey Cancer Center), the VCU
School of Pharmacy (to K.C.E.), and R01-CA-140416 from the
National Institutes of Health (to R.G.M.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Professor Shirley Taylor for assistance with
the cellular growth inhibition assay, Professor Matthew
Hartman and Professor John C. Hackett for helpful discussions,
and Professor Glen Kellogg for assistance in preparing this
manuscript.
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